Extracorporeal blood circulation (ECBC) has been employed in many important clinical situations such as renal dialysis and cardiopulmonary bypass. In the US alone, more than 15 million ECBC procedures are performed each year. All these applications require heparin anticoagulation to prevent clotting in the devices. However, systemic heparinization results in a high incidence of hemorrhagic complications. Heparin has been cited as the drug responsible for the most deaths. To avoid the bleeding complications associated with the use of heparin, protamine is administered at the conclusion of the ECBC procedure to reverse the anticoagulant activity of heparin. Unfortunately, intravenous administration of protamine is at times associated with serious, life-threatening adverse reactions. Recently it has been suggested that the administration of protamine may be a major cause of morbidity and mortality in patients undergoing cardiopulmonary bypass procedures. We have proposed an approach which would simultaneously prevent both heparin and protamine induced complications. The approach consists of placing a filter device containing immobilized protamine (termed as a protamine filter) at the distal end of an extracorporeal device where the blood re-enters the patient. The protamine filter would selectively remove heparin from the extracorporeal circuit, thereby eliminating heparin induced bleeding risks. the protamine filter would also confine protamine from entering the patient. Since protamine toxicity results either from the contact of protamine with tissue cells or from the circulating protamine-heparin complexes, the use of the protamine filter would minimize the toxic effects of protamine. A FIRST Award has been received from NIH to demonstrate the feasibility of the approach. During this grant period, significant progress has been made. The progress is reflected in the publication of 25 papers, 23 abstracts, and 4 patents. Thus far we have been able to: (i) develop a prototype protamine filter and demonstrate its improved blood compatibility; (ii) demonstrate the feasibility of the approach in removing heparin and preventing protamine-induced hemodynamic responses in animals; (iii) develop methods for filter sterilization, regeneration and storage, and also for preventing protamine leakage from the filter; (iv) characterize the adsorption characteristics and also establish a model to predict the behavior of the protamine filter; (v) develop a strip-type heparin assay, and also the first electrochemical heparin sensor; and (vi) develop a rapid colorimetric protamine titration method, and also a method for plasma protamine quantitation. Our specific aims in this proposal are: [1] to improve the chemistry for protamine immobilization; [2] to thoroughly evaluate the blood compatibility of the protamine filter; [3] to design highly effective filters with a target size as small as a single hemodialyzer cartridge but a capacity sufficient enough to remove heparin under cardiopulmonary bypass and hemodialysis conditions; [4] to test the filter ex vivo using sheep as the animal model; [5] to examine the feasibility of utilizing the protamine filter approach to prevent Type I protamine hypersensitivity in protamine-sensitized sheep.